Post on 27-Apr-2023
Decreased DGCR8 Expression and miRNA Dysregulationin Individuals with 22q11.2 Deletion SyndromeChantal Sellier1, Vicki J. Hwang2, Ravi Dandekar2, Blythe Durbin-Johnson3, Nicolas Charlet-Berguerand1,
Bradley P. Ander4,5, Frank R. Sharp4,5, Kathleen Angkustsiri4,6, Tony J. Simon4,7, Flora Tassone2,4*
1 Institute of Genetics and Molecular and Cellular Biology, University of Strasbourg, Strasbourg, France, 2 Department of Biochemistry and Molecular Medicine, University
of California Davis, Sacramento, California, United States of America, 3 Department of Public Health Sciences, UC Davis Medical Center, Sacramento, California, United
States of America, 4 MIND Institute, UC Davis Medical Center, Sacramento, California, United States of America, 5 Department of Neurology, UC Davis Medical Center,
Sacramento, California, United States of America, 6 Department of Pediatrics, UC Davis Medical Center, Sacramento, California, United States of America, 7 Department of
Psychiatry, UC Davis Medical Center, Sacramento, California, United States of America
Abstract
Deletion of the 1.5–3 Mb region of chromosome 22 at locus 11.2 gives rise to the chromosome 22q11.2 deletion syndrome(22q11DS), also known as DiGeorge and Velocardiofacial Syndromes. It is the most common micro-deletion disorder inhumans and one of the most common multiple malformation syndromes. The syndrome is characterized by a broadphenotype, whose characterization has expanded considerably within the last decade and includes many associatedfindings such as craniofacial anomalies (40%), conotruncal defects of the heart (CHD; 70–80%), hypocalcemia (20–60%), anda range of neurocognitive anomalies with high risk of schizophrenia, all with a broad phenotypic variability. Thesephenotypic features are believed to be the result of a change in the copy number or dosage of the genes located in thedeleted region. Despite this relatively clear genetic etiology, very little is known about which genes modulate phenotypicvariations in humans or if they are due to combinatorial effects of reduced dosage of multiple genes acting in concert. Here,we report on decreased expression levels of genes within the deletion region of chromosome 22, including DGCR8, inperipheral leukocytes derived from individuals with 22q11DS compared to healthy controls. Furthermore, we founddysregulated miRNA expression in individuals with 22q11DS, including miR-150, miR-194 and miR-185. We postulate this tobe related to DGCR8 haploinsufficiency as DGCR8 regulates miRNA biogenesis. Importantly we demonstrate that the level ofsome miRNAs correlates with brain measures, CHD and thyroid abnormalities, suggesting that the dysregulated miRNAsmay contribute to these phenotypes and/or represent relevant blood biomarkers of the disease in individuals with 22q11DS.
Citation: Sellier C, Hwang VJ, Dandekar R, Durbin-Johnson B, Charlet-Berguerand N, et al. (2014) Decreased DGCR8 Expression and miRNA Dysregulation inIndividuals with 22q11.2 Deletion Syndrome. PLoS ONE 9(8): e103884. doi:10.1371/journal.pone.0103884
Editor: Tao Cai, NIDCR/NIH, United States of America
Received September 9, 2013; Accepted July 8, 2014; Published August 1, 2014
Copyright: � 2014 Sellier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (Grant #R01HD042974 and UL1TR000002) and by Matteo’s Funds. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Dr. Flora Tassone has consulted with Novartis and Genentech.
* Email: ftassone@ucdavis.edu
Introduction
22q11.2 Deletion Syndrome (22q11DS) (OMIM #611867),
also known as DiGeorge (OMIM #188400) and Velocardiofacial
(OMIM #192430) syndromes, derives from the most common
chromosomal deletion associated with birth defects in humans and
it is estimated to occur in 1:4000 to 1:9700 live births [1–5].
Deletions are thought to arise from the mis-pairing of low copy
repeat (LCR) regions during cell division, resulting in a 3 Mb
deletion in 70–80% of individuals (between LCRs A-D) or to a
nested 1.5 Mb deletion in 15–30% of individuals (between LCRs
A–B) [6,7]. Atypical deletions within the 22q region are present in
the remainder of individuals [6].
Individuals with 22q11DS are characterized by a wide range of
clinical manifestations typically including abnormal development
of a variety of organs and structures (heart, palate, thyroid and
kidney), immunological anomalies, and neurological deficits that
lead to behavioral disorders and learning disabilities. 22q11DS is
also the second most common genetic cause of congenital heart
defects (CHDs) and cardiovascular malformations are present in
75% of patients. Conotruncal cardiac defects are the most
common type of CHDs in these individuals and include truncus
arteriosus, Tetralogy of Fallot, interrupted aortic arch, pulmonary
atresia, and ventricular septal defects. Additionally, 22q11DS is
the leading genetic cause of cleft palate that affects up to 42% of
individuals. Kidney defects are seen in up to one-third of these
individuals and can require life-long medical attention [8]. A high
prevalence of behavioral disorders has also been observed in
individuals with 22q11DS. Rates of Attention Deficit Hyperactiv-
ity Disorder (ADHD) vary from 52% in preteens to around 15–
30% across adulthood. Anxiety disorders are highly prevalent at
50–60% in childhood and somewhat less in adulthood [9]. Several
studies based on parent reports have indicated rates of 20–50% of
Autism Spectrum Disorders (ASDs) in children with 22q11DS
although smaller studies using gold standard diagnostic methods
found that no child met the criterion for ASD [10–12].
0.6–2% of schizophrenia cases have been attributed to the
22q11DS microdeletion and it is estimated that 30% of individuals
with 22q11DS will develop some type of schizophrenia later in
adolescence or adulthood [9,13–15]. With respect to neural
anomalies, individuals with 22q11DS show decreases in total and
PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e103884
regional brain volumes [16,17]. In particular, the hippocampus,
which is involved in memory and spatial processing, has been
strongly implicated in the pathophysiology of schizophrenia in the
general population and in 22q11DS [14,16,18–20]. Hippocampal
volume has also been linked to verbal IQ scores, which are
reduced in individuals with 22q11DS and have been found to
predict psychosis risk in that population [21–25].
Thirty genes are hemizygously deleted in the 1.5 Mb deletion
and up to 60 genes are hemizygously deleted in the 3 Mb deletion
[26]. However, it is not known if disease phenotypes arise from a
combinatorial effect of reduced dosage from multiple genes that
act together to lead to this diverse spectrum of phenotypes. The
majority of genes located in the chromosome 22 deletion region
have roles in RNA processing and signaling. Meechan et al. [27]
demonstrated a significant decrease in mRNA expression levels for
nine genes located within this region (Prodh2, Zdhhc8, Comt-mb,Tbx1, Ufd1l, Hira, Idd, Ranbp1, and T10) in the aortic arches
and heart of LgDel mouse embryos. Additionally, gene expression
levels of all genes located in the deletion region have been shown
to be decreased in brains of a 22q11DS mouse model when
compared to that of wild type (WT) mice [27–29].
Candidate genes for the CHD phenotype, such as TBX1, have
been highly touted; however, no pathogenic mutations have been
found [30–32]. Additionally, the presence of 22q11DS individuals
with CHD but without a TBX1 deletion suggests that TBX1 alone
does not lead to CHD [33].
One of the genes deleted in the majority of individuals with
22q11DS is the DiGeorge Critical Region Gene 8 (DGCR8) gene
which encodes a crucial component of the microprocessor
complex that contributes to microRNA (miRNA, miR) biogenesis
and therefore, to global gene regulation. miRNAs are ,22
nucleotide long and regulate gene expression, operating primarily
through post-transcriptional gene silencing by binding to their
target RNAs and are involved in many biological processes
including development, cell death and cell metabolism [34]. In
animals, the microprocessor complex processes mature miRNAs
to target specific mRNAs for translational repression [35]. The
Dgcr8+/2 mice shows behavioral and cognitive anomalies
including hyperactivity, abnormalities in sensorimotor gating,
and impaired spatial working memory consistent with altered
brain miRNA biogenesis due to Dgcr8 haploinsufficiency [29].
Indeed, Stark et al. showed that prefrontal cortex and hippocam-
pal miRNA levels were significantly downregulated (,20–70%)
and their mRNA target transcripts were shown to be significantly
dysregulated in a Df16+/2 mouse model [29]. Fenelon et al. [36]
demonstrated that Dgcr8+/2 mice have altered neuronal mor-
phology and synaptic properties indicative of the altered short-
term plasticity underlying cognitive dysfunction seen in individuals
with 22q11DS. Muscle specific Dgcr82/2 mice demonstrated
downregulation of a subset of mature cardiac enriched miRNAs,
specifically miR-1, miR-133a, and miR-208, and experienced
premature lethality within 2 months likely due to cardiac failure
[37]. It has also been reported that loss of miRNAs in the neural
crest (cells derived from form the pharyngeal arches in the
developing embryo that eventually mature into the palate, face,
and heart) lead to cardiac defects that are also observed in
individuals with 22q11DS [38]. Lastly, a recent report demon-
strated miRNA dysregulation in individuals with 22q11DS suggest
a role in the immunological, cardiac and hypocalcemic phenotypes
observed in this syndrome [39].
Here, we report on the decreased expression levels of several
genes, including DGCR8, within the 22q deletion region in
individuals with 22q11DS and compared to age matched controls.
Furthermore, we report on miRNA dysregulation and on the
potential contribution of specific miRNAs to multiple neurological
and structural phenotypes seen in these individuals.
Our findings support the hypothesis that alteration of the
miRNA landscape in individuals with 22q11DS contributes to the
observed neuronal cell dysfunction and clinical phenotypes.
Results
Human SubjectsDemographic information and clinical data are shown in
Table 1 for the subjects with 22q11DS. Clinical evaluation found
that sixteen subjects (53%) presented with CHD (including
Tetralogy of Fallot, atrial septal defects, polmonary atresia and
ventricular septal defects), 33% had hypocalcemia, 10% has renal
abnormalities, 16% had thyroid abnormalities and 20% had
moderate or severe seizure (Table 1).
Size Deletion AnalysisDeletion endpoints were verified using quantitative PCR
(qPCR) in 30 individuals with 22q11DS. 29 individuals had the
typical ,3 Mb deletion spanning PRODH-D22S936 while one
individual had the smaller ,1.5 Mb deletion spanning from
TUPLE1-ZNF74. A diagram depicting the deletion endpoints
observed in the individuals included in this study is shown in
Figure 1. qPCR values are shown in Table S1. Interestingly, the
individual with the ,1.5 Mb deletion had an IQ = 87 but
presented with severe symptoms, including multiple heart prob-
lems, ADHD, hypocalcemia and seizures (Table 1), supporting
the hypothesis of a minimal critical region for disease located
between LCR A-LCR B.
mRNA expression levels of genes mapping within thedeleted region
To determine if the hemizygous deletion resulted in reduced
expression of genes mapping within the deleted region, we
measured the gene expression levels in peripheral blood leukocytes
derived from individuals with 22q11DS of the following six genes:
catechol-O-methyltransferase; COMT, DiGeorge syndrome criti-
cal region gene 6; DGCR6, DiGeorge syndrome critical region
gene 8; DGCR8, zinc finger DHHC-type containing 8; ZDHHC8,
thioredoxin reductase 2; TXNRD2, and solute carrier family 25
member 1; SLC25A1 and of one gene, the Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) used as control. COMTencodes for a cytosolic enzyme that acts to degrade neuroactive
monoamines such as dopamine and it has been considered a
possible candidate gene for schizophrenia and other psychiatric
disorders [40,41]. DGCR6 encodes for a putative gonadal protein
that is involved with germ cell and gonadal development [42,43].
ZDHHC8 encodes for the palmitoyltransferase enzyme that is
responsible for posttranslational modifications [44]. ZDHHC8 has
been associated with susceptibility to schizophrenia and Mukai et
al. demonstrated that transfection of a ZDHHC8 carrying
construct into primary hippocampal neurons of Df(16)A+/2 mice
could recover dendritic spine density and growth to nearly WT
levels [45]. SLC25A1 and TXNRD2 encode for mitochondrial
proteins responsible for transporting citrate across the mitochon-
drial inner membrane and a thioredoxin reductase that plays a
large role in intracellular redox regulation, respectively [46,47].
mRNA expression levels for each gene were measured in 90
individuals with 22q11DS and 40 age matched controls by
quantitative real-time PCR (qRT-PCR). As expected, mRNA
expression analysis showed ,0.5 fold decrease in expression levels
in peripheral blood leukocytes of those with 22q11DS when
compared to controls for all six genes (Figure 2), although with
Altered miRNA Expression in 22q11DS
PLOS ONE | www.plosone.org 2 August 2014 | Volume 9 | Issue 8 | e103884
Ta
ble
1.
De
mo
gra
ph
ican
dcl
inic
ald
ata
of
the
ind
ivid
ual
sw
ith
22
q1
1D
sin
clu
de
din
the
stu
dy.
Ind
ivid
ua
lA
ge
Ge
nd
er
De
leti
on
siz
eT
yp
eo
fC
HD
aH
yp
oca
lce
mia
bS
eiz
ure
sdR
en
al
Ab
nb
Th
yro
idA
bn
b
Le
ftH
PC
Vo
lum
e(m
L)e
Rig
ht
HP
CV
olu
me
(mL
)e
Wh
ole
Bra
inV
olu
me
(mL
)fH
ea
dC
ircu
mfe
ren
ce
19
F3
Mb
02
00
01
.63
1.8
81
24
2.5
65
4.6
0
28
M3
Mb
00
20
21
.32
1.4
31
07
0.6
84
8.5
31
5M
3M
b0
00
00
1.7
51
.97
12
31
.16
54
.5
41
4M
3M
b0
00
00
1.6
1.6
71
35
3.0
15
9(N
/A)
58
M3
Mb
00
00
01
.67
1.7
51
29
4.1
15
2
61
0M
3M
bT
OF,
VaR
00
N/A
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1.2
91
.35
10
89
.06
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.75
(N/A
)
71
1F
3M
bT
A2
02
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9.5
89
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Mb
VR
00
00
1.4
71
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43
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53
91
1M
3M
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OF,
PA
20
00
N/A
N/A
11
88
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52
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10
15
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Mb
TO
F,V
SD0
00
01
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1.2
71
08
4.9
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/A
11
12
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Mb
VR
,B
AV
20
00
1.5
61
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7
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8M
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53
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02
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21
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41
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1&
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10
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/AN
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10
8.8
85
0.5
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11
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Mb
VSD
00
00
N/A
N/A
N/A
51
.5
19
11
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Mb
02
10
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/AN
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43
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45
7
20
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3M
bA
SD/V
SD0
00
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21
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21
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bV
R0
22
0N
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6
22
8M
3M
b0
00
20
N/A
N/A
N/A
52
.5
23
8M
3M
bT
OF
20
00
N/A
N/A
N/A
54
.6
24
11
F3
Mb
PD
A,
ASD
/VSD
,P
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3.5
25
5F
3M
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00
00
N/A
N/A
N/A
52
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26
4F
3M
b0
N/A
cN
/AN
/AN
/AN
/AN
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27
5M
3M
b0
00
N/A
N/A
N/A
N/A
N/A
51
28
8M
3M
b0
00
00
1.5
41
.66
12
79
.48
54
.6
Altered miRNA Expression in 22q11DS
PLOS ONE | www.plosone.org 3 August 2014 | Volume 9 | Issue 8 | e103884
some variation in levels. In addition to reduced mRNA expression
levels, DGCR8 protein expression levels, as measured by Western
blot analysis, were also reduced in individuals with 22q11DS
(Figure 3). As expected, no differences in GAPDH expression
levels were observed between 22q11.2DS and healthy controls
(Figure 2).
miRNA Expression LevelsTo investigate the presence of miRNA dysregulation as a result
of haploinsufficiency of the DGCR8 gene, we measured the
expression of selected miRNAs in 45 individuals, 30 with
22q11DS (same as those analyzed for deletion endpoints) and 15
typical developing control subjects (TD). The rationale for miRNA
selection was based on their role in CHD and to their association
with 22q11DS phenotypes observed in previous studies [29,39].
Seven miRNAs showed a differential expression between the two
groups (Figure 4). Of the differentially expressed miRNAs, miR-
185, miR-150, miR-194, and miR-363 were downregulated in
individuals with 22q11DS as compared to TD controls and miR-
208, miR-190, and miR-1 were upregulated. We also tested the
expression of two mirtrons, mi-877 and mi-1224, which are
miRNAs processed by the splicing machinery, and whose
biogenesis is therefore independent of DGCR8/DROSHA [48].
qRT-PCR analysis showed that no significant differences in
expression levels of mi-877 and mi-1224 (p = 0.843 and p = 0.491,
respectively) were present between the two groups (22q11DS,
n = 30; TD, n = 15) suggesting that mono-allelic expression of
DGCR8 specifically reduced the activity of DROSHA, without
affecting the splicing or the RNA polymerase II machinery.
Additionally, we measured the expression level of primary-miR-
324-5p and primary-miR-23b transcripts in the 30 individuals
with 22q11DS and found that they were significantly increased
(p,0.001 and p = 0.002 respectively) compared to those measured
in 15 TD individuals, while the expression level of the mature
miR-324-5p and miR-23b transcripts were decreased (p = 0.001
and p = 0.078 respectively) compared to those measured in TD
individuals, strongly suggesting that the observed reduced
expression levels of several miRNA are likely due to haploinsuffi-
ciency of DGCR8.
We then examined patient phenotypes to determine if the levels
of specific miRNA(s) were associated with clinical phenotypes
including brain volume, thyroid dysfunction, hypocalcemia,
seizure and CHD.
A significant inverse correlation between decreased miRNA
expression and increased brain volume was detected in the
22q11DS cohort. Specifically, six miRNAs (miR-185, miR-15b-
3p, miR-363, miR-324-5p, miR-361-5p, and miR-194) were
dysregulated in individuals with 22q11DS when examining left
hippocampal volume (Figure 5), and also with right hippocampal
volume (Figure 6), while the expression level of two miRNAs
(miR-361-5p, and miR-194) significantly decreased with increased
whole brain volume (Figure 7). In addition, our data showed that
miR-194 was significantly up-regulated by 2.13 fold in individuals
with 22q11DS who had thyroid dysfunction (p = 0.037), and by
2.02-fold in individuals with 22q11DS who had CHD (p = 0.012),
compared to unaffected subjects. No significant differences in
expression levels of any of the miRNAs analyzed were found when
individuals with 22q11DS were compared to controls for seizures,
renal abnormalities, head circumference and hypocalcemia.
Discussion
Mammalian development is sensitive to perturbation of gene
signaling such that either gain or loss of function of a single allele
Ta
ble
1.
Co
nt.
Ind
ivid
ua
lA
ge
Ge
nd
er
De
leti
on
siz
eT
yp
eo
fC
HD
aH
yp
oca
lce
mia
bS
eiz
ure
sdR
en
al
Ab
nb
Th
yro
idA
bn
b
Le
ftH
PC
Vo
lum
e(m
L)e
Rig
ht
HP
CV
olu
me
(mL
)e
Wh
ole
Bra
inV
olu
me
(mL
)fH
ea
dC
ircu
mfe
ren
ce
29
3F
3M
bA
SD/V
SD0
00
01
.21
1.6
19
57
2.9
74
8.5
30
9M
1.5
Mb
IAA
,A
SD/V
SD,
mild
aort
icst
en
osi
s2
1&
20
01
.78
1.9
11
20
6.4
85
3
aT
OF
=T
etr
alo
gy
of
Fallo
t;P
A=
Pu
lmo
nar
yA
tre
sia;
VSD
=V
en
tric
ula
rSe
pta
lD
efe
ct;
TA
=T
run
cus
Art
eri
osu
s;V
R=
Vas
cula
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ing
;B
AV
=B
icu
spid
Ao
rtic
Val
ve,
VaR
=V
alve
Re
pla
cem
en
t;A
SD=
Atr
ial
Sep
tal
De
fect
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AA
=A
ort
icA
rch
Ab
no
rmal
itie
s;IA
A=
Inte
rru
pte
dao
rtic
arch
.b
0=
No
;2
=Y
es.
cN
/A=
Dat
an
ot
avai
lab
le.
d0
=N
o;
1=
Hyp
oca
lce
mic
seiz
ure
s;2
=Y
es.
eA
vera
ge
hip
po
cam
pal
volu
me
for
TD
ind
ivid
ual
sis
2m
L.f A
vera
ge
wh
ole
bra
invo
lum
efo
rT
Din
div
idu
als
is1
20
0m
L.H
PC
=h
ipp
oca
mp
us.
Ab
n=
abn
orm
alit
y.d
oi:1
0.1
37
1/j
ou
rnal
.po
ne
.01
03
88
4.t
00
1
Altered miRNA Expression in 22q11DS
PLOS ONE | www.plosone.org 4 August 2014 | Volume 9 | Issue 8 | e103884
of genes mapping within the deleted region can affect development
and function. However, additional factors can play a role and
account for the broad variation in the phenotypes we observed in
individuals with 22q11DS. It is not clear if altered gene dosage of a
number of genes, or if any specific ones, lead to the observed
phenotypes by means of a cumulative effect, or if other
mechanisms play a role. The impact of several deleted genes is
what likely may determine the overall 22q11DS phenotype and it
has become increasingly clear that the 22q11DS symptoms most
likely arise from a combination of factors including gene
expression of genes inside and outside of the deletion region
(modifier genes) and/or from the individual’s genetic background
[49]. Inconsistent phenotypes have been reported in siblings and
monozygotic twins that have concordant 22q deletions reflecting
environmental effects [50–53]. We characterized the deletion
endpoints of the subjects included in our study and found that the
majority of individuals (n = 29) had the same 3 Mb deletion;
however, one individual had a smaller, nested deletion spanning
from TUPLE1 to the ZNF74 gene. Individuals with interstitial
deletions and severe phenotype have been previously reported in
the literature. McQuade et al. [54] described a patient with a 750
Kb deletion encompassing COMT and TBX1. He presented with
many of the typical 22q11DS symptoms such as cleft palate, facial
features typical for 22q11DS, low IQ, schizophrenia, OCD, and
developmental delay. Individuals carrying an atypical deletion and
yet presenting with the characteristic features of 22q11DS, suggest
that a minimal critical region is sufficient to lead to disease
phenotypes.
Altered dosage of several genes in the 22q deletion region is
hypothesized to correlate with phenotypic variation. In the present
study, we demonstrated approximately a 50% reduction in the
expression levels of several candidate genes in the deleted region
(COMT, DGCR6, DGCR8, ZDHHC8, TXNRD2 and SLC25A1)
in peripheral blood leukocytes from young and adolescent
participants with 22q11DS, and demonstrate a ,50% decrease
in mRNA levels when compared to controls. One of these genes,
DGCR8, plays an important role in miRNA biogenesis by
encoding for a crucial component of the microprocessor complex
that processes primary microRNA (pri-miRNAs) transcripts to
mature microRNAs [29,53].
Indeed, as a consequence of DGCR8 haploinsufficiency and as
observed in the 22q deletion mouse model, we demonstrate
miRNA dysregulation in peripheral blood leukocytes derived from
individuals with 22q11DS [29,55]. The lack of significant
difference observed between individuals with 22q11DS compared
to TD in the expression levels of two mirtrons, whose biogenesis
depends on alternative, non-canonical, miRNA biogenesis path-
ways via splicing, in addition to higher expression levels of primary
miRNA and of lower of mature miRNA of those found to be
dysregulated in individuals with 22q11DS compared to TD, are
supportive of the hypothesis that altered miRNA expression
pattern observed in this study likely results from the hemizygous
DGCR8 expression [56]. These results also show that transcription
and processing of miRNAs are not globally altered in 22q11DS
and provide compelling evidence implicating microRNA-mediat-
ed dysregulation in 22q11DS.
Our findings confirm previous reports [29,39,55] and further
suggest that haploinsufficiency of DGCR8 could alter the miRNA
expression landscape and contribute to the wide clinical pheno-
type, including cognitive, neurocognitive, psychiatric disorders and
cardiac disease, observed in 22q11DS. Importantly, we demon-
strate, for the first time, that altered miRNA levels correlates with
brain measures which is intriguing in light of the recent report of
hippocampus-dependent spatial learning, memory, and social
behavior deficits and decreased hippocampal adult neurogenesis
observed in the Dgcr8/mouse model [57].
Thus, we propose a model where hemizygous deletion of
DGCR8 leads to a decrease in microprocessor efficiency and to a
dysregulation of miRNA expression, which ultimately contributes
to the clinical phenotypes seen in individuals with 22q11DS
(Figure 8).
miRNAs play an important role in the regulation and
modulation of many biological functions and their altered
expression has been observed in a variety of human diseases
including cardiovascular and heart diseases [58].
A number of miRNAs including miR-194, miR-361, miR-150
and miR-185 were found to be dysregulated in 22q11DS. In
Figure 1. Diagram of the 22q11.2 deletion region. Schematic overview of chromosome 22 and the deletion endpoints characterized in theparticipants of this study using qPCR. Individuals 1 to 29 had a deletion between PRODH and D22S936. One individual had the deletion locatedbetween the TUPLE1 and ZNF74 genes. Gene used, order of genes, LCRs A–D, and the common 3 Mb and 1.5 Mb deletion are indicated. Thin blacklines indicate deleted region, solid black thick lines indicate chromosomal regions present in two copies, and gray boxes indicate uncertain locationof the deletion breakpoints. Each numbered chromosome represents one individual.doi:10.1371/journal.pone.0103884.g001
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particular, highly significant lower expression levels were observed
for miR-194, miR-361 and miR-185 in individuals with 22q11DS
across all of our phenotypic neuronal measures. Down-regulation
of miR-194 was observed in both the prefrontal cortex and
hippocampus in the 22q11DS mouse model, suggests a potential
role in the development of the central nervous system [29]. miR-
194 is also highly enriched in kidney, differentially expressed in
renal carcinoma and can affect cell migration [59]. Renal
abnormalities are often observed in 22q11DS; however they were
present only in a few individuals in our group (Table 1). More
importantly, we observed a statistically significant difference in its
expression levels within the 22q11 group between subjects with
CHD compared to those without, particularly in relation to the
recent findings of miR-194 involvement in acute myocardial
infarction in individuals experiencing heart failure [60].
The observed down-regulation of miR-185 was expected as it is
located within the deleted region of chromosome 22 and it was
found to be significantly down-regulated across many of the
neurological measures. miR-185 is implicated in many neurolog-
ical disorders leading to hypotonic infants and it has also been
suggested as a key player in neuronal development. In this study
down-regulation of miR-185 correlated with brain volume, in
agreement with findings in both the prefrontal cortex and the
hippocampus of the mouse model of 22q11DS [29]. miR-185
Figure 2. Gene expression levels. Box plots showing the relative transcript expression levels for six genes mapping within the deleted region andof the GAPDH gene, used as control. Total RNA was isolated from 90 individuals with 22q11DS (22q) and from 40 typical developing age-matchedcontrols (TD). Error bars indicate standard errors. mRNA levels were normalized to the reference Glucuronidase gene. Respective p-values are shown.doi:10.1371/journal.pone.0103884.g002
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contributes to dendritic and spine development deficits in
hippocampus of the Df(16)A+/2 mouse model [55]. This and
previous research showing the presence of miR-185 at the
synapses [61,62], suggest that miR-185 may have a role in neural
function and constitute a key gene regulator in 22q11DS.
In this study we also observed that decreased miR-324-5p
expression levels correlated with increased left and right hippo-
campal volume; interestingly miR-324-5p promotes neuronal
differentiation and it may contributes to the development of
defined neuronal subtypes [63,64].
Importantly the miRNAs, which expression was found altered
in this study, are expressed in both peripheral blood leukocytes
and brain tissue and for several of them the expression was found
to be altered in psychiatric disorders including schizophrenia and
depression [65–68].
Several miRNAs have been implicated in cardiogenesis
and heart development including miR-1, miR208 and miR-190
[69–71]. In the presented study we observed an increased
expression of miR-208 and miR-190 in 22q11DS compared to
controls; interestingly a significant up-regulation of these miRNAs
were reported in patients with myocardial infarction [69]
suggesting their contributing role in cardiac disease. Thus, it is
possible that upregulation of these miRNA in patients with
22q11DS is secondary to cardiac alterations and likely overcome
the decrease processing efficiency due to DGCR8 haploinsuffi-
ciency.
Finally, we demonstrate that several miRNAs including miR-
361 and miR 194 are downregulated in blood, suggesting that
dysregulation of the miRNA biogenesis in 22q11DS occurs in
blood and that the degree of dysregulation can correlate with
psychiatric, neurocognitive and immunological features of
22q11DS. Several reports have indicated the usefulness of using
blood for miRNA expression studies and have demonstrated
altered expression of blood and brain miRNAs involved in brain
Figure 3. DGCR8 expression levels. a) box plots showing a decreased DGCR8 mRNA levels in subjects with 22q11DS compared to TD and b)DGCR8 protein expression levels measured by Western blot analysis shows a significant decrease in 22q11DS individuals when compared to TDcontrols. c) Representative Western blot showing decreased DGCR8 expression.doi:10.1371/journal.pone.0103884.g003
Figure 4. Altered miRNA expression in 22q11DS. Relative miRNA levels are shown for 7 miRNAs that demonstrated differential expressionlevels when comparing 30 individuals with 22q11DS and 15 TD individuals. miRNA levels were normalized to the reference U6 snRNA. Circlesrepresent observed data. Horizontal lines represent geometric means. Respective p-values are shown.doi:10.1371/journal.pone.0103884.g004
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plasticity and maturation associated to schizophrenia and ASD
[72–75]. These studies suggest that lymphocytes could reflect, or
partially share, the molecular phenotype of neural cells and could
therefore be used in studies of psychiatric disorders. This is
important since the identification of altered miRNAs expressed in
blood could be used as biomarkers to identify potential therapeutic
targets and to monitor the response to potential therapeutic
approaches of the disease.
We expected that some miRNAs, including miR-1 and miR-
208, although differentially expressed in subjects with 22q11DS
compared to TD, were dysregulated between individuals with
22q11DS with CHD compared to those without CHD, but we did
not detect any difference in levels between the two groups. This
may reflect the age of the individuals studied as previous studies
indicating a critical role for microRNAs in maintaining cardiac
function were performed on prenatal mice [37]. The observed lack
of association could also be due to the modest number of 22q11DS
subjects with specific types of CHD, which is one of the limitations
of this study.
Elucidating miRNA dysregulation in 22q11DS will promote
understanding of the contribution of miRNAs in neurocognitive,
cardiac, behavioral and other common diseases that demonstrate
differential miRNA expression such as cancer. While it is well
known that individual miRNAs have important roles in neuronal
and cardiovascular function and development, the in vivoimplications are less well understood. Here, we demonstrate that
miRNAs are associated with a variety of clinical abnormalities and
importantly, that they can be detected in blood suggesting a
potential use for circulating miRNAs as non-invasive biomarkers.
That circulating miRNA can serve as novel diagnostic markers
and that these miRNAs can be delivered to recipient cells, where
they can regulate translation of target genes, has been proposed for
a wide range of cardiovascular disease [76].
The documentation of molecular markers or abnormalities that
underlie the phenotypic involvement of psychiatric, cardiac, or
neurological dysfunction will lead to earlier treatment for the
problems seen in childhood and adolescence in individuals with
22q11DS.
Our findings, in addition to several studies in the mouse models
of 22q11DS, provide compelling evidence that the syndrome
results in abnormal processing of miRNAs. Our findings are in
agreement with those recently reported by de la Morena et al. but
extended into a wider age range [39]. We also reported
correlations between miRNA dysregulation and key phenotypic
characteristics of 22q11DS including those involving the CNS and
thyroid abnormalities and more importantly demonstrated an
effect of decreased miRNA levels in brain measures.
In conclusion, we show that miRNA dysregulation in 22q11DS
is due to DGCR8 haploinsufficiency and that this may contribute
to significant dysregulation of their target mRNAs ultimately
leading to the clinical phenotype observed in these individuals.
However, it remains to be determined if the miRNA dysregulation
Figure 5. Observed correlation between dysregulated miRNAs and left hippocampal volume. Graphs showing a correlation betweenmiRNA expression levels and left hippocampal volume within the 22q11DS group. Open circles represent individuals with 22q11DS; the linerepresents line of best fit. P-values given are for correlations.doi:10.1371/journal.pone.0103884.g005
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Figure 6. Observed correlation between dysregulated miRNAs and right hippocampal volume. Graphs showing a significant correlationbetween miRNA expression levels and right hippocampal volume within the individuals with 22q11DS are shown. Open circles represent individualswith 22q11DS; the line represents line of best fit. P-values given are for each miRNA.doi:10.1371/journal.pone.0103884.g006
Figure 7. Observed correlation between dysregulated miRNAs and whole brain volume. Graphs showing a significant correlationbetween miRNA expression levels and whole brain volume within the individuals with 22q11DS are shown. Open circles represent individuals with22q11DS; the line represents line of best fit. P-values given are for each miRNA.doi:10.1371/journal.pone.0103884.g007
Altered miRNA Expression in 22q11DS
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here described is also present in brain tissue and therefore playing
a key role in the neurological phenotype seen in 22q11DS. Further
studies are also necessary to determine if miRNA profiles along
with other biomarkers and cognitive testing could potentially
provide a more comprehensive and early diagnosis of 22q11DS.
Finally, the ultimate challenge will be to identify and validate
the target genes that are affected by those microRNAs that are
found dysregulated in 22q11DS and to characterize the pathways
of involvement in a much more comprehensive manner in order to
improve our understanding of how alterations in microRNA-
mediated genetic networks can contribute to the pathophysiology
of 22q11DS. Although further studies are required to assess the
mechanism of action of miRNA acting as gene modulators,
ultimately this information will be of relevance for the developing
of novel therapeutic strategies.
Materials and Methods
Human ParticipantsParticipants were recruited for behavioral and developmental
assessments at the UC Davis Medical Investigation of Neurode-
velopmental Disorders (MIND) Institute located in Sacramento,
California, under written consent from the next of kin, caretakers,
or guardians on the behalf of the minors/children participants and
according to a UC Davis Institutional Review Board (IRB)
approved protocol. The diagnosis of 22q11DS was performed by
FISH analysis using the TUPLE1 probe. Age- and gender-
matched typically developing children (TD) were used as controls
and the inclusion criteria was FSIQ .85, normal ADHD and
ADS scores [77] and normal social functioning [78]. All
participants were examined by a developmental behavioral
pediatrician or child and adolescent psychiatrist. Physical condi-
tions (cardiac, neurologic, immunologic, renal, etc.) were obtained
from the parents and when available, medical record abstraction.
Medical conditions were rated on a severity scale described in
Table 1. Molecular measures were determined based on sample
availability. Peripheral blood was collected in EDTA blood
collection tubes and in Tempus tubes (Applied Biosystems, Foster
City, CA) and DNA and totRNA respectively isolated for further
analysis. For mRNA expression analysis, we examined a total of
130 individuals, 90 individuals with 22q11DS including 40 males
and 50 females, known to have 22q11DS by FISH and, 40 TD
individuals including 19 males and 21 females. Age range of
participants was 7 to 21 years (mean age is 13.163.5 years for TD
and 10.962.6 years for 22q11DS individuals). For protein
expression analysis, we examined a total of 31 individuals (17
22q11DS, including 14 males and 3 females, and 14 TD, including
5 males and 9 females) where the age range of participants is 7–15
years (mean age is 11.462.6 (22q) and 10.562.4 (TD)). In a
subgroup of them (30 individuals with 22q11DS), miRNA analysis
and deletion endpoint analyses were both carried out. Specifically,
for miRNA expression analyses, we examined a total of 45
individuals (30 22q11DS, including 19 males and 11 females, and
15 TD, including 9 males and 6 females), where the age range of
participants was 4–15 years and the mean age is 10.764.5 (TD)
and 8.963.4 (22q11DS) years for which totRNA (from PAX tubes;
Qiagen, Valencia, CA) containing miRNAs was available. For
deletion endpoint analyses, we examined a total of 30 individuals
with 22q11DS, including 19 males and 11 females where the age
range is 4–15 years (mean 8.963.4 years). There was no statistical
Figure 8. Proposed model of miRNA dysregulation in 22q11DS. Decreased levels of free DGCR8 results in dysregulated expression of maturemiRNAs, ultimately leading to development of the clinical phenotypes associated with 22q11DS.doi:10.1371/journal.pone.0103884.g008
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difference in age range between the two groups for any of the
analyses.
Deletion Endpoint Analysis by qPCRqPCR and quantification of deletion endpoints was analyzed
using methods described in Weksberg et al. [79] with the following
minor modifications. Samples from 30 subjects with 22q11DS (19
male and 11 female) and 9 typically developing (TD) subjects (16
male and 8 female) were analyzed. Primers were designed using
Primer Express v2.0 (Applied Biosystems, Carlsbad, CA) and
followed the recommended primer design guidelines. Target
assays, D22S181, DGCR6, PRODH, TUPLE1, ZNF74, LZTR1,D22S936, and VPREB1, and reference assays, HEM3 and
G6PDH, were used as described in Weksberg et al. [79]. The
sequences of additional primers used including TBX1, DGCR8,SCARF2, and SHGC-2421 were as follow: Tbx1-F:GTCCGAAATCAAGCGAGTGAGTA; Tbx1-R: GTGGAGG-
AAACCCTGGTCAAC; DGCR8-F: GCATGTGTTCCTTCT-
GCTCTGAT; DGCR8-R: CAGGACGCACTGAGGAGGTA-
GT; SCARF2-F: CATGGTGTAGGGCCAGTCTATCC; SC-ARF2-R: CAGCCATTCGCACTTTAGAGAAA; Shgc-2421-F:TCATGTGGGTGCTGGTACAT; Shgc-2421-R: AGCTTCA-
GGCTCTCCAGACA.
Reactions were performed using FastStart Universal SYBR
Green Master Mix (Roche, Applied Science, Indianapolis, IN)
(which includes the internal reference (ROX)), forward and
reverse primers at final concentrations of 800 nM for the
22q11DS target primers and 400 nM for the reference primers,
and 10 ng of genomic DNA. The qPCR reactions were run using
the Applied Biosystems 7900HT FAST real-time PCR system with
2 min at 50uC, 10 min at 95uC followed by 40 cycles of 15 sec at
95uC and 60 sec at 60uC. qPCR data was analyzed using the
comparative Ct method after data normalization [79].
Quantitative Real Time-PCR for gene expression analysisof genes in the deleted region
Total RNA was isolated from Tempus tubes (Applied Biosys-
tems, Carlsbad, CA) from blood drawn from 130 individuals, 90
with 22q11DS (22q) and 40 TD individuals using standard
procedures. RNA was quantified with a NanoDrop 1000 UV/VIS
spectrophotometer (Thermo Fisher Scientific, Inc., Waltham,
MA). mRNA expression levels were measured using a quantita-
tive-fluorescence reverse transcription polymerase chain reaction
(qRT-PCR) method and analyzed on a 7900HT FAST real-time
PCR system (Applied Biosystems, Carlsbad, CA). cDNA synthesis
and real-time PCR were performed following previous methods
[80]. Assay on Demand Gene Expression assays (Applied
Biosystems, Carlsbad, CA) were used for each gene.
Western BlotLymphocytes pellets isolated from 2 ml whole blood and
available from 31 individuals (17 22q11DS and 14 TD) were
washed twice using a PBS (137 mmol/L NaCl, 2.7 mmol/L KCl,
4.3 mmol/L Na2KH2PO4, pH 7.4) (Gibco, Carlsbad, CA) and
Calbiochem protease inhibitor cocktail III (Millipore, Billerica,
MA) and then resuspended in MPER lysis buffer (Pierce,
Rockford, IL) with Roche complete protease inhibitor tablet
(Roche Diagnostics, Indianapolis, IN) and Calbiochem cocktail
III. Cell lysis was performed by sonication using the LabQuake
(Thermo Fisher Scientific, Waltham, MA). The Quick Start
Bovine Serum Albumin Standard set kit (BioRad Laboratories,
Hercules, CA) was used according to manufacturer’s instructions
for quantification of protein concentration. Cell-extracted proteins
were separated using electrophoresis at 80 V, at RT, on a 8–13%
Criterion Tris-HCl gel (BioRad Laboratories, Hercules, CA) in
running buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1%
SDS, pH 8.3). Proteins were transferred for 3 hours at 250 mA, at
room temperature, using Criterion Cell Blotter System (BioRad,
Hercules, CA) onto nitrocellulose membranes (BioRad Laborato-
ries, Hercules, CA) in 25 mmol/L Tris, 192 mmol/L glycine, and
20% methanol. Ponceau staining of the nitrocellulose membrane
and Coommassie Blue (Invitrogen, Carlsbad, CA) staining of the
gel verified efficient transfer of proteins. Following the transfer,
membranes were blocked with 5% nonfat dry milk in T-TBS
(100 mmol/L Tris, 150 mmol/L NaCl, 0.1% polyoxyethylene
sorbitan monolaurate), and then incubated with secondary
antibody (1:20,000 mouse anti-GAPDH (Millipore, Billerica,
MA) and 1:400 rabbit anti-DGCR8 (Proteintech, Chicago, IL).
Super Signal West Dura substrate (Pierce, Rockford, IL) was used
for the detection of antibodies. AlphaView gel analysis software
was used to analyze band intensities (Protein Simple, Santa Clara,
CA).
miRNA Extraction, RT-PCR and target analysisTotal RNA from 45 individuals (30 22q11DS and 15 TD) was
isolated from PAX tubes (Qiaen, Valencia, CA) and/or from
cell pellets using Trizol (Life Technologies, Carlsbad, CA).
cDNAs were generated using the miScript II RT Kit (Qiagen,
Valencia, CA) for quantification of miRNAs or the Transcriptor
High Fidelity cDNA Synthesis Kit (Roche Diagnostics, India-
napolis, IN) for quantification of mRNAs. qPCR of miRNAs
was performed using the miScript Primer Assay (Qiagen,
Valencia, CA) and miScript Sybr Green PCR Kit (Qiagen,
Valencia, CA) in a LightCycler 480 (Roche Diagnostics,
Indianapolis, IN) with 15 min at 94uC followed by 50 cycles
of 15 s at 94uC, 20 s at 55uC, and 20 s at 72uC. U6 snRNA was
used as standard.
Statistical AnalysisFor mRNA and protein expression, a two-sample t-test was
used to test for differences in mean expression between the
22q11DS and TD groups for each gene. P-values were adjusted
using the Bonferroni correction. Two sample t-tests were used to
compare miRNA expression between TD and all 22q11DS
participants, or between 22q11DS participants with and without
a given clinical phenotype. miRNA expression was compared
using one-way ANOVA followed by Tukey HSD pairwise
comparisons between TD participants, those with 22q11DS
and CHD, and those with 22q11DS but without CHD. miRNA
expression was correlated with brain measurements using linear
regression. miRNA data were log transformed prior to analysis;
results are reported on the original scale of the data as fold
changes. Analyses were conducted using R, version 3.0.0 (R Core
Team, 2013).
Supporting Information
Table S1 Fold copy number change by qPCR. (DKCt) for
individuals with 22q11DS (n = 30) and TD individuals (n = 3) for
three assays (PRODH, DGCR8, and D22S936) are shown. DKCt
values for TUPLE1 and ZNF74 for one individual (#30) with an
atypical deletion are shown to better characterize the deletion size.
DKCt values of 060.35 indicate an equal ratio of the target and
reference (normal) while values of 2160.35 indicate a hemizygous
deletion.
(DOCX)
Altered miRNA Expression in 22q11DS
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Acknowledgments
We would like to thank Nimrah Choudhary for technical assistance and
Hiu Tung Tang for technical assistance and graphic work.
This work is dedicated to the memory of Matteo.
Author Contributions
Conceived and designed the experiments: VJH RD CS NCB FT.
Performed the experiments: VJH RD CS BPA KA. Analyzed the data:
VJH RD BDJ CS NCB FRS TJS FT. Contributed reagents/materials/
analysis tools: CS NCB BPA FRS TJS. Wrote the paper: VJH FT.
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